The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill-defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D-glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [3H]-thymidine incorporation and cell-cycle regulatory protein expression levels compared with 5 mM D-glucose or 25 mM L-glucose. In addition, high glucose increased transforming growth factor-β1 (TGF-β1) mRNA and protein expression levels. High glucose-induced cell-cycle regulatory protein expression levels and [3H]-thymidine incorporation, which were inhibited by TGF-β1 siRNA transfection and TGF-β1 neutralizing antibody treatment. High glucose-induced phosphorylation of protein kinase C (PKC), p44/42 mitogen-activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time-dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10−6 M; bisindolylmaleimide I, 10−6 M), LY 294002 (PI3 kinase inhibitor, 10−6 M), Akt inhibitor (10−5 M), PD 98059 (p44/42 MAPKs inhibitor, 10−5 M), SB 203580 (p38 MAPK inhibitor, 10−6 M), and rapamycin (mTOR inhibitor, 10−8 M) blocked the high glucose-induced cellular proliferation and TGF-β1 protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF-β1 expression via Ca2+/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010 © 2010 Wiley-Liss, Inc.